The family of CDC25s phosphatases represents important regulators of the cell cycle that are required for activation of the CDK-cyclin complexes to promote the cell cycle progression. The CDC25A and CDC25B phosphatases are classical proto-oncogenes, and alteration in their function promotes oncogenic transformation in vivo. CDC25 phosphatases are frequently overexpressed in a variety of cancers, which correlates with poor prognosis. Importantly, downregulation of CDC25s by small interfering RNAs has been shown to inhibit tumor growth validating CDC25s as very important therapeutic targets. Targeting protein phosphatases using small molecules is generally difficult. Despite numerous efforts, well validated and non-covalent inhibitors of CDC25s have not been described to date. The active site of these enzymes is very shallow and lacks well defined binding pocket. Furthermore, CDC25s share highly reactive active site cysteine with other protein tyrosine phosphatases hampering screening and design efforts. In consequence, CDC25s proved difficult to target by small molecules and one of the most potent inhibitors described to date are quinone derivatives which most likely represent covalent inhibitors of phosphatases. In this project, we propose a novel approach to develop small molecules targeting CDC25B phosphatase by inhibiting the protein-protein interaction between CDC25B and CDK2/CycA complex. The CDK2/CycA represents a natural substrate of CDC25B, which is recognized via an interface that is distant from the active site. We propose to identify small molecule compounds binding to CDC25B at this interface to inhibit the interaction with CDK2/Cyclin A. Our proposal represents a very innovative approach to identify potent and non- covalent inhibitors of CDC25 phosphatases. We have developed high quality assay for assessing the protein- protein interaction between CDC25B and CDK2/CycA and will use high-throughput screening to identify potent small molecule inhibitors of this interaction. The screen will be carried out in the CCG center at the University of Michigan. Subsequently we will employ a panel of orthogonal assays to eliminate false positives and biophysical and structural biology methods to select direct non-covalent inhibitors. The activities of identified compounds will be assessed in cellular experiments. The long term goal of our project is to develop potent cell- cycle inhibitors as novel anti-cancer agents.